Cationic lipids and transfection methods

ABSTRACT

The present invention relates in part to novel cationic lipids and their use, e.g., in delivering nucleic acids to cells.

PRIORITY

The present application is a continuation of U.S. application Ser. No.16/930,901, filed Jul. 16, 2020 which is a continuation of U.S.application Ser. No. 16/746,279, filed Jan. 17, 2020 (now U.S. Pat. No.10,752,576), which is a continuation of U.S. application Ser. No.16/660,317, filed Oct. 22, 2019 (now U.S. Pat. No. 10,611,722), which isa continuation of U.S. application Ser. No. 16/526,621, filed Jul. 30,2019 (now U.S. Pat. No. 10,501,404), the content of each of which ishereby incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates, in part, to various novel lipids,including methods, compositions, and products for delivering nucleicacids to cells.

BACKGROUND

Lipid-based materials, such as liposomes, are used as biologicalcarriers for pharmaceutical and other biological applications, e.g., tointroduce agents into cultured cell lines. Lipids are commonly used todeliver nucleic acids to cells in vitro under low-serum or serum-freeconditions, for instance in transfection. However, serum componentsinhibit the activity of many lipids, limiting their use in the presenceof serum, both in vitro and in vivo.

Improved lipid delivery systems, e.g., to achieve higher levels oftransfection both in vitro and in vivo, are desirable. In particular,lipid delivery systems that are active in the presence of serum areneeded. Improved levels of transfection will allow the treatment ofdisease states for which higher levels of expression than are currentlyachievable with lipid delivery systems are needed for therapeuticeffect. Alternatively, higher transfection levels will allow for use ofsmaller amounts of material to achieve comparable expression levels,thereby decreasing potential toxicities and decreasing cost.

There is a need for novel lipids, lipid-like materials, and lipid-baseddelivery systems in the art.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to new lipids that find use,inter alia, in improved delivery of biological payloads, e.g. nucleicacids, to cells.

In aspects, the present invention relates to a compound of Formula (I)

where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

In embodiments, n is 1-12, or 2-12, or 1-10, or 2-10, or 1-8, or 2-8, or2-6.

In embodiments, the present invention relates to compound (i):

In embodiments, the present invention relates to compound (ii):

In embodiments, the present invention relates to compound (iii):

In embodiments, the present invention relates to compound (iv):

In embodiments, the present invention relates to compound (v):

In embodiments, the present invention relates to compound (vi):

In embodiments, the present invention relates to compound (vii):

In embodiments, the present invention relates to compound (viii):

In embodiments, the present compounds (e.g. of Formula I) are componentsof a pharmaceutical composition and/or a lipid aggregate and/or a lipidcarrier and/or a lipid nucleic-acid complex and/or a liposome and/or alipid nanoparticle.

In embodiments, the present compounds (e.g. of Formula I) are componentsof a pharmaceutical composition and/or a lipid aggregate and/or a lipidcarrier and/or a lipid nucleic-acid complex and/or a liposome and/or alipid nanoparticle which does not require an additional or helper lipid.

In embodiments, the present compounds (e.g. of Formula I) are componentsof a pharmaceutical composition and/or a lipid aggregate and/or a lipidcarrier and/or a lipid nucleic-acid complex and/or a liposome and/or alipid nanoparticle which comprises a nucleic acid, such as DNA (e.g.,without limitation, a plasmid, cosmid, phage, recombinant virus or othervector) or RNA (e.g., without limitation, an siRNA, micro-RNA (miRNA),long non-coding RNA (lncRNA), an in vitro transcribed RNA, a syntheticRNA, and/or an mRNA, in each case that comprises one or morenon-canonical nucleotides that confer stability, avoid degradation byone or more nucleases, and/or avoid substantial cellular toxicity, ordoes not comprise a non-canonical nucleotide).

In aspects, the present invention relates to a method for transfecting acell with a nucleic acid, comprising contacting the cell with a complexof the nucleic acid and a compound described herein (e.g. of Formula I),where the complex of the nucleic acid and the compound described herein(e.g. of Formula I) is optionally formed prior to contact with the cell.

In embodiments, the transfection method provides at least one of thefollowing characteristics: (a) high transfection efficiency, (b) highlevel of endosomal escape, (c) serum-resistance, (d) low toxicityeffects, (e) high level of protein expression, (f) transfectability invarious cell types, and (g) transfectability without additional lipidsor reagents for transfection, e.g. relative to a method of transfectinga cell with complex of the nucleic acid and DOTMA, DODMA, DOTAP, DODAP,DOPE, cholesterol, LIPOFECTIN (cationic liposome formulation),LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000(cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposomeformulation), and combinations thereof.

The details of the invention are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, illustrative methods and materials are now described.Other features, objects, and advantages of the invention will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

Any aspect or embodiment disclosed herein can be combined with any otheraspect or embodiment as disclosed herein.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 depicts primary human epidermal keratinocytes cultured in a24-well plate, and transfected with 400 ng per well of in vitrotranscribed RNA encoding green fluorescent protein (GFP) complexed withthe indicated lipid and with the indicated mass ratio of lipid to RNA.Complexation was performed in DMEM, and transfections were performed in100% fetal bovine serum (FBS). Images were taken eight hours followingtransfection.

FIG. 2 depicts the experiment of FIG. 1 , with fluorescence measured atthe indicated time points following transfection using DHDLinS.

FIG. 3 depicts the results of an experiment conducted as in FIG. 1 , butwith the indicated amounts of RNA (in nanograms) and the indicatedlipid-to-RNA mass ratios (in micrograms of lipid per microgram of RNA).Images were taken 16 hours following transfection. As shown in thefigure, all RNA amounts and lipid-to-RNA mass ratios tested yielded afluorescent signal.

FIG. 4 depicts the results of an experiment conducted as in FIG. 1 , butwith human peripheral blood mononuclear cells (hPBMCs) instead ofkeratinocytes. Images were taken 16 hours following transfection.“LF3000” indicates cells transfected with LIPOFECTAMINE 3000 (cationicliposome formulation) commercial transfection reagent. “Neg.” indicatesun-transfected cells.

FIG. 5A depicts the results of an experiment conducted as in FIG. 4 ,but with a confluent layer of primary human epidermal keratinocytesinstead of hPBMCs. Images were taken 24 hours following transfection.

FIG. 5B depicts the experiment of FIG. 5A, shown at highermagnification.

FIG. 6A depicts the results of an experiment conducted as in FIG. 4 ,but with primary human adult dermal fibroblasts instead of hPBMCs.Images were taken 16 hours following transfection.

FIG. 6B depicts the experiment of FIG. 6A, shown at highermagnification.

FIG. 7 depicts the results of an experiment conducted as in FIG. 3 , butwith DHDLinS purified by extraction with acetone as described in Example5.

FIG. 8 depicts primary human epidermal keratinocytes cultured in a24-well plate, and transfected with 100 ng per well of in vitrotranscribed RNA encoding green fluorescent protein (GFP) complexed withthe compounds of Formula I, where n is as indicated. Images were takeneight hours following transfection.

FIG. 9A depicts the measured 500 MHz proton NMR spectrum of DHDLinS indeuterated chloroform.

FIG. 9B depicts DHDLinS.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery of novellipids that, inter alia, demonstrate superior abilities to supportdelivery of nucleic acids to cells, e.g. during transfection. Thepresent invention provides such compositions, methods of making thecompositions, and methods of using the compositions to introduce nucleicacids into cells, including for the treatment of diseases.

Compounds

In aspects, the present invention relates to a compound of Formula (I)

where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

In embodiments, n is 1-14, or 1-12, or 1-10, or 1-8, or 1-6, or 1-4, or1-2, or 2-14, or 2-12, or 2-10, or 2-8, or 2-6, or 2-4, or 4-14, or4-12, or 4-10, or 4-8, or 4-6, or 6-14, or 6-12, or 6-10, or 6-8, or8-14, or 8-12, or 8-10, or 10-14, or 10-12.

In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 3.In embodiments, n is 4. In embodiments, n is 5. In embodiments, n is 6.In embodiments, n is 7. In embodiments, n is 8. In embodiments, n is 9.In embodiments, n is 10. In embodiments, n is 11. In embodiments, n is12. In embodiments, n is 13. In embodiments, n is 14. In embodiments, nis 15.

In embodiments, the present invention relates to compound (i):

In embodiments, the present invention relates to compound (ii):

In embodiments, the present invention relates to compound (iii):

In embodiments, the present invention relates to compound (iv):

In embodiments, the present invention relates to compound (v):

In embodiments, the present invention relates to compound (vi):

In embodiments, the present invention relates to compound (vii):

In embodiments, the present invention relates to compound (viii):

In embodiments, the present invention relates to a pharmaceuticalcomposition and/or a lipid aggregate and/or a lipid carrier and/or alipid nucleic-acid complex and/or a liposome and/or a lipid nanoparticlewhich comprises a compound described herein (e.g. of Formula I).

In embodiments, the pharmaceutical composition and/or lipid aggregateand/or lipid carrier and/or lipid nucleic-acid complex and/or liposomeand/or lipid nanoparticle is in any physical form including, e. g.,lipid nanoparticles, liposomes, micelles, interleaved bilayers, etc.

In embodiments, the pharmaceutical composition and/or lipid aggregateand/or lipid carrier is a liposome. In embodiments, the liposome is alarge unilamellar vesicle (LUV), multilamellar vesicle (MLV) or smallunilamellar vesicle (SUV). In embodiments, the liposome has a diameterup to about 50 to 80 nm. In embodiments, the liposome has a diameter ofgreater than about 80 to 1000 nm, or larger. In embodiments, theliposome has a diameter of about 50 to 1000 nm, e.g. about 200 nm orless. Size indicates the size (diameter) of the particles formed. Sizedistribution may be determined using quasi-elastic light scattering(QELS) on a Nicomp Model 370 sub-micron particle sizer.

In embodiments, the compound (e.g. of Formula I), and/or pharmaceuticalcomposition and/or lipid aggregate and/or lipid carrier and/or lipidnucleic-acid complex and/or liposome and/or lipid nanoparticlecomprising the compound (e.g. of Formula I), is soluble in an alcohol(e.g. ethyl alcohol) at room temperature (e.g. about 20-25° C.) and/orat low temperatures (e.g. about 0° C., or about −10° C., or about −20°C., or about −30° C., or about −40° C., or about −50° C., or about −60°C., or about −70° C., or about −80° C.).

In certain embodiments, the present invention relates to methods andcompositions for producing lipid-encapsulated nucleic acid particles inwhich nucleic acids are encapsulated within a lipid layer. Such nucleicacid-lipid particles, including, without limitation incorporating RNAs,can be characterized using a variety of biophysical parametersincluding: drug to lipid ratio; encapsulation efficiency; and particlesize. High drug to lipid ratios, high encapsulation efficiency, goodnuclease resistance and serum stability and controllable particle size,generally less than 200 nm in diameter can, in certain situations, bedesirable (without limitation).

Nucleic acid to lipid ratio can refer to the amount of nucleic acid in adefined volume of preparation divided by the amount of lipid in the samevolume. This may be on a mole per mole basis, or on a weight per weightbasis, or on a weight per mole basis, or on a mole per weight basis. Forfinal, administration-ready formulations, the nucleic acid to lipidratio may be calculated after dialysis, chromatography and/or enzyme(e.g., nuclease) digestion has been employed to remove as much externalnucleic acid as possible.

Encapsulation efficiency can refer to the drug (including nucleic acid)to lipid ratio of the starting mixture divided by the drug (includingnucleic acid) to lipid ratio of the final, administration competentformulation. This can be a measure of relative efficiency. For a measureof absolute efficiency, the total amount of nucleic acid added to thestarting mixture that ends up in the administration competentformulation, can also be calculated. The amount of lipid lost during theformulation process may also be calculated. Efficiency can be used as ameasure of the wastage and expense of the formulation.

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles have utility for delivery of macromolecules andother compounds into cells. In embodiments, the present compounds (e.g.of Formula I) and/or pharmaceutical compositions and/or lipid aggregatesand/or lipid carriers and/or lipid nucleic-acid complexes and/orliposomes and/or lipid nanoparticles have utility for delivery ofnucleic acids into cells.

In embodiments, there is provided a method for transfecting a cell witha nucleic acid, comprising contacting the cell with a complex of thenucleic acid and a present compound (e.g. of Formula I) and/orpharmaceutical composition and/or lipid aggregate and/or lipid carrierand/or lipid nucleic-acid complex and/or liposome and/or lipidnanoparticle. In embodiments, the complex of the nucleic acid and thepresent compound (e.g. of Formula I) and/or pharmaceutical compositionand/or lipid aggregate and/or lipid carrier and/or lipid nucleic-acidcomplex and/or liposome and/or lipid nanoparticle is formed prior tocontact with the cell.

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles encapsulate nucleic acids with high-efficiency,and/or have high drug to lipid ratios, and/or protect the encapsulatednucleic acid from degradation and/or clearance in serum, and/or aresuitable for systemic delivery, and/or provide intracellular delivery ofthe encapsulated nucleic acid. In addition, in embodiments, the presentcompounds (e.g. of Formula I) and/or pharmaceutical compositions and/orlipid aggregates and/or lipid carriers and/or lipid nucleic-acidcomplexes and/or liposomes and/or lipid nanoparticles are well-toleratedand provide an adequate therapeutic index, such that patient treatmentat an effective dose of the nucleic acid is not associated withsignificant toxicity and/or unacceptable risk to the patient.

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles are polycationic. In embodiments, the presentcompounds (e.g. of Formula I) and/or pharmaceutical compositions and/orlipid aggregates and/or lipid carriers and/or lipid nucleic-acidcomplexes and/or liposomes and/or lipid nanoparticles form stablecomplexes with various anionic macromolecules, such as polyanions, suchas nucleic acids, such as RNA or DNA. In embodiments, the presentcompounds (e.g. of Formula I) and/or pharmaceutical compositions and/orlipid aggregates and/or lipid carriers and/or lipid nucleic-acidcomplexes and/or liposomes and/or lipid nanoparticles, have theproperty, when dispersed in water, of forming lipid aggregates whichstrongly, via their cationic portion, associate with polyanions. Inembodiments, by modulating the amount of cationic charges relative tothe anionic compound, for example by using an excess of cationic chargesrelative to the anionic compound, the polyanion-lipid complexes may beadsorbed on cell membranes, thereby facilitating uptake of the desiredcompound by the cells.

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles mediate one or more of (i) compacting a nucleic acidpayload to be delivered, which may protect it from nuclease degradationand/or may enhance receptor-mediated uptake, (ii) improving associationwith negatively-charged cellular membranes, which may be modulated bygiving the complexes a positive charge, (iii) promoting fusion withendosomal membranes, which may facilitate the release of complexes fromendosomal compartments, and (iv) enhancing transport from the cytoplasmto the nucleus.

In embodiments, the present invention relates to the present compounds(e.g. of Formula I) and/or pharmaceutical compositions and/or lipidaggregates and/or lipid carriers and/or lipid nucleic-acid complexesand/or liposomes and/or lipid nanoparticles for transfection, or methodsof transfection, which have a high transfection efficiency. Inembodiments, the transfection efficiency is measured by assaying apercentage of cells that are transfected compared to the entirepopulation, during a transfection protocol. In various embodiments, thetransfection efficiency of the present compositions and methods isgreater than about 30%, or greater than about 40%, or greater than about50%, or greater than about 60%, or greater than about 70%, or greaterthan about 80%, or greater than about 90%, or greater than about 95%. Invarious embodiments, the transfection efficiency of the presentcompositions and methods is greater than the transfection efficiency ofcommercially available products (e.g. LIPOFECTIN (cationic liposomeformulation), LIPOFECTAMINE (cationic liposome formulation),LIPOFECTAMINE 2000 (cationic liposome formulation), LIPOFECTAMINE 3000(cationic liposome formulation) (Life Technologies)). In variousembodiments, the transfection efficiency of the present compositions andmethods is about 1.1-fold, or about 1.5-fold, or about 2-fold, or about5-fold, or about 10-fold, or about 15-fold, or about 20-fold, or about30-fold, or about 50-fold, or greater than about 50-fold greater thanthe transfection efficiency of commercially available products (e.g.LIPOFECTIN (cationic liposome formulation), LIPOFECTAMINE (cationicliposome formulation), LIPOFECTAMINE 2000 (cationic liposomeformulation), LIPOFECTAMINE 3000 (cationic liposome formulation) (LifeTechnologies)).

In embodiments, the present invention relates to the present compounds(e.g. of Formula I) and/or pharmaceutical compositions and/or lipidaggregates and/or lipid carriers for transfection, or methods oftransfection, which permit a high level of endosomal escape. In variousembodiments, the endosomal escape of the present compositions andmethods is greater than the endosomal escape of commercially availableproducts (e.g. LIPOFECTIN (cationic liposome formulation), LIPOFECTAMINE(cationic liposome formulation), LIPOFECTAMINE 2000 (cationic liposomeformulation), LIPOFECTAMINE 3000 (cationic liposome formulation) (LifeTechnologies)). In various embodiments, the endosomal escape of thepresent compositions and methods is about 5-fold, or 10-fold, or15-fold, or 20-fold, or 30-fold greater than the endosomal escape ofcommercially available products (e.g. LIPOFECTIN (cationic liposomeformulation), LIPOFECTAMINE (cationic liposome formulation),LIPOFECTAMINE 2000 (cationic liposome formulation), LIPOFECTAMINE 3000(cationic liposome formulation) (Life Technologies)).

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles are serum-resistant. In embodiments, the presentcompounds (e.g. of Formula I) and/or pharmaceutical compositions and/orlipid aggregates and/or lipid carriers and/or lipid nucleic-acidcomplexes and/or liposomes and/or lipid nanoparticles are substantiallystable in serum. In embodiments, the present transfection methods canfunction in the presence of serum and/or do not require seruminactivation and/or media changes. In embodiments, the stability inserum and/or serum-resistance is measurable via in vitro assays known inthe art. Transfection efficiency in varying amounts of serum may be usedto assess the ability to transfect a macromolecule (e.g., withoutlimitation, DNA or RNA), optionally in comparison to commerciallyavailable products (e.g. LIPOFECTIN (cationic liposome formulation),LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000(cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposomeformulation) (Life Technologies)).

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles for transfection, or methods of transfection, havelow or reduced toxicity effects. In embodiments, the present compounds(e.g. of Formula I) and/or pharmaceutical compositions and/or lipidaggregates and/or lipid carriers and/or lipid nucleic-acid complexesand/or liposomes and/or lipid nanoparticles for transfection, or methodsof transfection, have reduced toxicity effects as compared tocommercially available products (e.g. LIPOFECTIN (cationic liposomeformulation), LIPOFECTAMINE (cationic liposome formulation),LIPOFECTAMINE 2000 (cationic liposome formulation), LIPOFECTAMINE 3000(cationic liposome formulation) (Life Technologies)). In variousembodiments, the present compositions and methods allow for cells havinggreater than about 50%, or about 60%, or about 70%, or about 80%, orabout 90%, or about 95% viability after transfection. In variousembodiments, the present compositions and methods allow for cells havingabout 1.1-fold, or about 1.5-fold, or about 2-fold, or about 5-fold, orabout 10-fold, or about 15-fold, or about 20-fold, or about 30-foldgreater viability after transfection, as compared to commerciallyavailable products (e.g. LIPOFECTIN (cationic liposome formulation),LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000(cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposomeformulation) (Life Technologies)). In embodiments, toxicity effectsinclude disruption of cell morphology and/or viability or deregulationof one or more genes.

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles for transfection, or methods of transfection, permita high level of protein expression from the nucleic acid (e.g. DNA orRNA) being transfected. In various embodiments, the protein expressionof the present compositions and methods is greater than about 30%, orgreater than about 40%, or greater than about 50%, or greater than about60%, or greater than about 70%, or greater than about 80%, or greaterthan about 90%, or greater than about 95% more than in un-transfectedcells and/or cells contacted with naked nucleic acid. In variousembodiments, the resultant protein expression of the presentcompositions and methods is greater than the resultant proteinexpression of commercially available products (e.g. LIPOFECTIN (cationicliposome formulation), LIPOFECTAMINE (cationic liposome formulation),LIPOFECTAMINE 2000 (cationic liposome formulation), LIPOFECTAMINE 3000(cationic liposome formulation) (Life Technologies)). In variousembodiments, the resultant protein expression of the presentcompositions and methods is about 1.1-fold, or about 1.5-fold, or about2-fold, or about 5-fold, or about 10-fold, or about 15-fold, or about20-fold, or about 30-fold, or about 50-fold, or greater than about50-fold greater than the resultant protein expression of commerciallyavailable products (e.g. LIPOFECTIN (cationic liposome formulation),LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000(cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposomeformulation) (Life Technologies)).

In embodiments, the present invention relates to the present compounds(e.g. of Formula I) and/or pharmaceutical compositions and/or lipidaggregates and/or lipid carriers and/or lipid nucleic-acid complexesand/or liposomes and/or lipid nanoparticles for transfection, or methodsof transfection, which allow for transfection, including efficienttransfection as described herein, in various cell types. In embodiments,the present compounds (e.g. of Formula I) and/or pharmaceuticalcompositions and/or lipid aggregates and/or lipid carriers fortransfection, or methods of transfection, allow for transfection,including efficient transfection as described herein, in establishedcell lines, hard-to-transfect cells, primary cells, stem cells, andblood cells. In embodiments, the cell type is a keratinocyte, afibroblast, a PBMC, or a dendritic cell.

In embodiments, a present compound (e.g. of Formula I), pharmaceuticalcomposition and/or a lipid aggregate and/or a lipid carrier and/or lipidnucleic-acid complex and/or liposome and/or lipid nanoparticle issuitable for transfection or delivery of compounds to target cells,either in vitro or in vivo.

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles for transfection, or methods of transfection, do notrequire additional reagents for transfection, e.g. PLUS™ Reagent (DNApre-complexation reagent) (Life Technologies).

In embodiments, the present compounds (e.g. of Formula I) are componentsof a pharmaceutical composition and/or a lipid aggregate and/or a lipidcarrier and/or lipid nucleic-acid complex and/or liposome and/or lipidnanoparticle which does not require an additional or helper lipid, e.g.for efficient transfection. In embodiments, the pharmaceuticalcomposition and/or a lipid aggregate and/or a lipid carrier and/or lipidnucleic-acid complex and/or liposome and/or lipid nanoparticle does notrequire one or more of: DOPE, DOPC, cholesterol, and a polyethyleneglycol (PEG)-modified lipid (inclusive, without limitation, of aPEGylated PE phospholipid, PC phospholipid, and/or cholesterol), e.g.for efficient transfection.

In embodiments, the present compounds (e.g. of Formula I) are componentsof a pharmaceutical composition and/or a lipid aggregate and/or a lipidcarrier and/or lipid nucleic-acid complex and/or liposome and/or lipidnanoparticle that further comprises an additional or helper lipid.

In embodiments, the additional or helper lipid is selected from one ormore of the following categories: cationic lipids; anionic lipids;neutral lipids; multi-valent charged lipids; and zwitterionic lipids. Insome embodiments, a cationic lipid may be used to facilitate acharge-charge interaction with nucleic acids.

In embodiments, the additional or helper lipid is a neutral lipid. Inembodiments, the neutral lipid is dioleoylphosphatidylethanolamine(DOPE), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), or cholesterol.In embodiments, cholesterol is derived from plant sources. In otherembodiments, cholesterol is derived from animal, fungal, bacterial orarchaeal sources.

In embodiments, the additional or helper lipid is a cationic lipid. Inembodiments, the cationic lipid isN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), or1,2-dioleoyl-3-dimethylammonium-propane (DODAP).

In embodiments, one or more of the phospholipids 18:0 PC, 18:1 PC, 18:2PC, DMPC, DSPE, DOPE, 18:2 PE, DMPE, or a combination thereof are usedas helper lipids. In embodiments, the additional or helper lipid isDOTMA and DOPE, optionally in a ratio of about 1:1. In embodiments, theadditional or helper lipid is DHDOS and DOPE, optionally in a ratio ofabout 1:1.

In embodiments, the additional or helper lipid is a commerciallyavailable product (e.g. LIPOFECTIN (cationic liposome formulation),LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000(cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposomeformulation) (Life Technologies)).

In embodiments, the additional or helper lipid is a compound having theFormula (A):

where, R1 and R4 are straight-chain alkenyl having 17 carbon atoms; R2and R5 are —(CH2)p-NH2 where p is 1-4; I is 1-10; and Xa is aphysiologically acceptable anion.

In one embodiment, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles include one or more polyethylene glycol (PEG)chains, optionally selected from PEG200, PEG300, PEG400, PEG600, PEG800,PEG1000, PEG1500, PEG2000, PEG3000, and PEG4000. In embodiments, the PEGis PEG2000. In embodiments, the present compounds (e.g. of Formula I)and/or pharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles include1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or a derivativethereof. In one embodiment, the present compounds (e.g. of Formula I)and/or pharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles comprise1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG); in another embodiment, the present compounds(e.g. of Formula I) and/or pharmaceutical compositions and/or lipidaggregates and/or lipid carriers and/or lipid nucleic-acid complexesand/or liposomes and/or lipid nanoparticles comprise1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DMPE-PEG); in yet another embodiment, the presentcompounds (e.g. of Formula I) and/or pharmaceutical compositions and/orlipid aggregates and/or lipid carriers and/or lipid nucleic-acidcomplexes and/or liposomes and/or lipid nanoparticles comprise1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG).In further embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles comprise a mixture of PEGylated lipids and/or freePEG chains.

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles comprise one or more ofN-(carbonyl-ethoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE),fully hydrogenated phosphatidylcholine, cholesterol, LIPOFECTAMINE 2000(cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposomeformulation), a cationic lipid, a polycationic lipid, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethyleneglycol)-5000] (FA-MPEG5000-DSPE).

In one embodiment, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles comprise about 3.2 mg/mLN-(carbonyl-ethoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE),about 9.6 mg/mL fully hydrogenated phosphatidylcholine, about 3.2 mg/mLcholesterol, about 2 mg/mL ammonium sulfate, and histidine as a buffer,with about 0.27 mg/mL1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethyleneglycol)-5000] (FA-MPEG5000-DSPE). In another embodiment, the nucleicacids are complexed by combining 1 μL of LIPOFECTAMINE 3000 (cationicliposome formulation) per about 1 μg of nucleic acid and incubating atroom temperature for at least about 5 minutes. In one embodiment, theLIPOFECTAMINE 3000 (cationic liposome formulation) is a solutioncomprising a lipid at a concentration of about 1 mg/mL. In embodiments,nucleic acids are encapsulated by combining about 1 μg, or about 2 μg,or about 5 μg, or about 10 μg of the present compounds (e.g. of FormulaI) and/or pharmaceutical compositions and/or lipid aggregates and/orlipid carriers and/or lipid nucleic-acid complexes and/or liposomesand/or lipid nanoparticles per about 1 μg of nucleic acid and incubatingat room temperature for about 5 minutes or longer than about 5 minutes.

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles comprise one or more nanoparticles. In oneembodiment, the nanoparticle is a polymeric nanoparticle. In variousembodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles comprise one or more of a diblock copolymer, atriblock copolymer, a tetrablock copolymer, and a multiblock copolymer.In various embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles comprise one or more of polymeric nanoparticlescomprising a polyethylene glycol (PEG)-modified polylactic acid (PLA)diblock copolymer (PLA-PEG), PEG-polypropylene glycol-PEG-modifiedPLA-tetrablock copolymer (PLA-PEG-PPG-PEG), and Poly(lactic-co-glycolicacid) copolymer. In another embodiment, the present compounds (e.g. ofFormula I) and/or pharmaceutical compositions and/or lipid aggregatesand/or lipid carriers and/or lipid nucleic-acid complexes and/orliposomes and/or lipid nanoparticles comprise a statistical, or analternating, or a periodic copolymer, or any other sort of polymer.

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles comprise one or more lipids that are described inWO/2000/027795, the entire contents of which are hereby incorporatedherein by reference.

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles comprises Polybrene™ (hexadimethrine bromide) asdescribed in U.S. Pat. No. 5,627,159, the entire contents of which areincorporated herein by reference.

In various embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles comprise one or more polymers. Examples of polymerinclude hexadimethrine bromide (Polybrene™), DEAE-Dextran, protamine,protamine sulfate, poly-L-lysine, or poly-D-lysine. These polymers maybe used in combination with cationic lipids to result in synergisticeffects on uptake by cells, stability of the present compounds (e.g. ofFormula I) and/or pharmaceutical compositions and/or lipid aggregatesand/or lipid carriers and/or lipid nucleic-acid complexes and/orliposomes and/or lipid nanoparticles, including serum stability (e.g.,stability in vivo), endosomal escape, cell viability, and proteinexpression.

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles are suitable for associating with a nucleic acid,inclusive of, for instance, include any oligonucleotide orpolynucleotide.

In embodiments, nucleic acids are fully encapsulated within the presentcompounds (e.g. of Formula I) and/or pharmaceutical compositions and/orlipid aggregates and/or lipid carriers and/or lipid nucleic-acidcomplexes and/or liposomes and/or lipid nanoparticles. In otherembodiments, nucleic acids are partially encapsulated within the presentcompounds (e.g. of Formula I) and/or pharmaceutical compositions and/orlipid aggregates and/or lipid carriers and/or lipid nucleic-acidcomplexes and/or liposomes and/or lipid nanoparticles. In still otherembodiments, nucleic acids and the present compounds (e.g. of Formula I)and/or pharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles are both present with no encapsulation of thenucleic acids within the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles.

Fully encapsulated can indicate that the nucleic acid in the presentcompounds (e.g. of Formula I) and/or pharmaceutical compositions and/orlipid aggregates and/or lipid carriers and/or lipid nucleic-acidcomplexes and/or liposomes and/or lipid nanoparticles is notsignificantly degraded after exposure to serum or a nuclease assay thatwould significantly degrade free nucleic acids. In embodiments, lessthan about 25% of particle nucleic acid is degraded in a treatment thatwould normally degrade about 100% of free nucleic acid. In embodiments,less than about 10% or less than about 5% of the particle nucleic acidis degraded.

Extent of encapsulation may be determined by an Oligreen assay. Oligreenis an ultra-sensitive fluorescent nucleic acid stain for quantitatingoligonucleotides and single-stranded DNA in solution (available fromInvitrogen Corporation, Carlsbad, CA).

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles are serum stable and/or they do not rapidlydecompose into their component parts upon in vivo administration.

In embodiments, the present compounds (e.g. of Formula I) and/orpharmaceutical compositions and/or lipid aggregates and/or lipidcarriers and/or lipid nucleic-acid complexes and/or liposomes and/orlipid nanoparticles are complexed with a nucleic acid (e.g. DNA or RNA)in a ratio, which may depend on the target cell type, generally rangingfrom about 1:16 to about 25:1 ng lipid:ng DNA or RNA. Illustrativelipid:DNA or RNA ratios are from about 1:1 to about 10:1, e.g. about1:1, or about 2:1, or about 3:1, or about 4:1 or about 5:1, or about6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1.

In embodiments, additional parameters such as nucleic acidconcentration, buffer type and concentration, etc., are selected toachieve a desired transfection efficiency, e.g., high transfectionefficiency.

In embodiments, the nucleic acid is selected from RNA or DNA.

In embodiments, the DNA is a plasmid, cosmid, phage, recombinant virusor other vector. In embodiments, a vector (or plasmid) refers todiscrete elements that are used to, for example, introduce heterologousnucleic acid into cells for expression or replication thereof. Inembodiments, the vectors can remain episomal or can be designed toeffect integration of a gene or portion thereof into a chromosome of thegenome. Also contemplated are vectors that are artificial chromosomes,such as yeast artificial chromosomes and mammalian artificialchromosomes. Included, without limitation, are vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments (e.g. expression vectors). Thus, a vector can referto a recombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the DNA. Appropriatevectors can include, without limitation, those that are replicable ineukaryotic cells and/or prokaryotic cells and those that remain episomalor those that integrate into the host cell genome.

In embodiments, the nucleic acid is an RNA, messenger RNA (mRNA), asmall interfering RNA (siRNA), micro RNA (miRNA), long non-coding RNA(lncRNA), antisense oligonucleotide, ribozyme, plasmid, immunestimulating nucleic acid, antisense, antagomir, antimir, microRNA mimic,supermir, U1 adaptor, or aptamer.

In embodiments, the RNA is a synthetic RNA. In embodiments, the RNA is achemically synthesized RNA. In embodiments, the RNA is an in vitrotranscribed RNA.

In embodiments, the synthetic RNA (inclusive, without limitation, ofmRNA) does not comprise a non-canonical nucleotide. In embodiments, thesynthetic RNA (inclusive, without limitation of mRNA) comprises one ormore non-canonical nucleotides. In embodiments, the one or morenon-canonical nucleotides is selected from 2-thiouridine, 5-azauridine,pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine,5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine,5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine,5-ethoxyuridine, 5-ethoxypseudouridine, 5-hydroxymethyluridine,5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine,5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine,5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine,5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine,4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine,4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine,4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine,4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine,4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine,pseudoisocytidine, N4-methylcytidine, N4-aminocytidine,N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine,5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine,5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine,5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine,5-methylpseudoisocytidine, 5-aminopseudoisocytidine,5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine,N4-methylpseudoisocytidine, 2-thio-5-azacytidine,2-thiopseudoisocytidine, 2-thio-N4-methylcytidine,2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine,2-thio-5-methylcytidine, 2-thio-5-aminocytidine,2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine,2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine,2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine,2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine,2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine,N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine,N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine,N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine,N4-methyl-5-aminopseudoisocytidine,N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine,N4-aminopseudoisocytidine, N4-amino-5-methylcytidine,N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine,N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine,N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine,N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine,N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine,N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine,N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine,N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine,N4-hydroxy-5-methylpseudoisocytidine,N4-hydroxy-5-aminopseudoisocytidine,N4-hydroxy-5-hydroxypseudoisocytidine,2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine,2-thio-N4-methyl-5-hydroxycytidine,2-thio-N4-methyl-5-methyl-5-azacytidine,2-thio-N4-methyl-5-amino-5-azacytidine,2-thio-N4-methyl-5-hydroxy-5-azacytidine,2-thio-N4-methyl-5-methylpseudoisocytidine,2-thio-N4-methyl-5-aminopseudoisocytidine,2-thio-N4-methyl-5-hydroxypseudoisocytidine,2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine,2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine,2-thio-N4-amino-5-hydroxycytidine,2-thio-N4-amino-5-methyl-5-azacytidine,2-thio-N4-amino-5-amino-5-azacytidine,2-thio-N4-amino-5-hydroxy-5-azacytidine,2-thio-N4-amino-5-methylpseudoisocytidine,2-thio-N4-amino-5-aminopseudoisocytidine,2-thio-N4-amino-5-hydroxypseudoisocytidine,2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine,2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine,2-thio-N4-hydroxy-5-hydroxycytidine,2-thio-N4-hydroxy-5-methyl-5-azacytidine,2-thio-N4-hydroxy-5-amino-5-azacytidine,2-thio-N4-hydroxy-5-hydroxy-5-azacytidine,2-thio-N4-hydroxy-5-methylpseudoisocytidine,2-thio-N4-hydroxy-5-aminopseudoisocytidine,2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine,N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine,8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine,7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine,N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine,N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine,N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine,N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine,8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine,7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.

In embodiments, the compound, pharmaceutical composition, or lipidaggregate described herein is complexed with or associates with anucleic acid (e.g. DNA or RNA, e.g. mRNA) and the nucleic acid encodes aprotein of interest. In embodiments, the protein of interest is asoluble protein. In embodiments, the protein of interest is one or moreof a reprogramming protein and a gene-editing protein.

This invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1: Synthesis of Linoleoyl Chloride (1)

Oxalyl chloride (47.0 mL, 555 mmol) was added to a solution of linoleicacid (70.0 g, 250 mmol) in 580 mL anhydrous methylene chloride at 0° C.under N₂ atmosphere. The reaction was warmed to room temperature andstirred vigorously for 24 hours. Solvent and oxalyl chloride wereremoved under reduced pressure to yield linoleoyl chloride as a brownoil, which was used without further purification.

Example 2: Synthesis of N1,N4-dilinoleoyl-diaminobutane (2)

A solution of 1,4-diaminobutane (0.428 g, 4.86 mmol) and triethylamine(2.03 mL, 14.6 mmol) in 1 mL of anhydrous methylene chloride was slowlyadded to a solution of linoleoyl chloride (2.98 g, 10.0 mmol) in 30 mLof anhydrous methylene chloride in an ice bath at 0° C. The reactionmixture was stirred vigorously with a magnetic stir bar. After additionwas complete, the ice bath was removed and the mixture was stirred atroom temperature for 2.5 days. The reaction was cooled to 4° C., and awhite solid precipitated from the solution. The excess linoleoylchloride was removed by vacuum filtration. The precipitate was washedtwice with 10 mL of methylene chloride. The mother liquor wasconcentrated and more product precipitated. This precipitate wasfiltered and combined with the previous precipitate. The resulting solidwas vacuum dried for 4 hours. A total of 1.9 g of a white solid of thedesired product, N¹,N⁴-dilinoleoyl-diaminobutane, was obtained.

Example 3: Synthesis of N1,N4-dilinoleyl-diaminobutane (3)

Lithium aluminum hydride (0.6 g, 95%, 16 mmol) was carefully added to asuspension of N¹,N⁴-dilinoleoyl-diaminobutane (1.8 g, 2.9 mmol) in 50 mLanhydrous diethyl ether at 0° C. After addition was complete, the icebath was removed. The reaction mixture was warmed slowly to roomtemperature and then heated gently to reflux with an appropriatecondensing device and stirred for 12 hours. The reaction mixture wascooled and quenched carefully at 0° C. with 5 mL of water. The diethylether was removed under reduced pressure, and the reaction mixture wasdried under vacuum. The dried reaction mixture was extracted three timeswith 25 mL of isopropyl alcohol at 80° C. The isopropyl alcohol wasremoved to yield 1.6 g of oily colorless N¹,N⁴-dilinoleyl-diaminobutane.

Example 4: Synthesis ofN1,N4-dilinoleyl-N1,N4-di-[2-hydroxy-3-(N-phthalamido)propyl]-diaminobutane(4)

Diisopropylethylamine (1.15 mL, 12.0 mmol) was added to a suspension ofN¹,N⁴-dilinoleyl-diaminobutane (1.6 g, 2.7 mmol) andN-(2,3-epoxypropyl)-phthalimide (1.6 g, 7.9 mmol) in 12 mL of dryN,N-dimethylformamide. After purging with nitrogen, the reaction mixturewas sealed in a round-bottom flask and heated to around 90° C. for 24hours. N,N-dimethylformamide and diisopropylethylamine were removed anda yellow oil was obtained. Synthesis was continued without additionalpurification.

Example 5: Synthesis ofN1,N4-dilinoleyl-N1,N4-di-(2-hydroxy-3-aminopropyl)-diaminobutane (5)

The entire crude oil ofN¹,N⁴-dilinoleyl-N¹,N⁴-di-[2-hydroxy-3-(N-phthalamido)propyl]-diaminobutanewas dissolved in 25 mL of anhydrous ethanol. Hydrazine (0.5 mL, 64-65%aq., 10.3 mmol) was added at room temperature. With an appropriatecondensing device, the reaction mixture was heated to reflux. The oilbath was set to 85° C. After 15 minutes, a white solid precipitated fromthe solution. The reaction mixture was stirred at reflux for 4 hoursbefore being cooled to −20° C. The white solid was removed by gravityfiltration. The residue was washed twice with cold ethanol. The combinedethanol solution was concentrated and dried overnight under vacuum. Thecrude product was extracted with acetone. The combined acetone solutionwas concentrated and dried overnight under vacuum. 1.0 g of an oil,N¹,N⁴-dilinoleyl-N¹,N⁴-di-(2-hydroxy-3-aminopropyl)-diaminobutane(referred to herein as DHDLinS, see FIG. 9B), was obtained. The protonNMR spectrum of a 1-2 mg sample of DHDLinS in 0.6 mL deuteratedchloroform was measured on a 500 MHz Varian (nova instrument (FIG. 9A).

Example 6: Synthesis of Lipids

The following compounds were synthesized by the methods of Examples 1through 5 using the corresponding amine:

-   N¹,N⁴-dilinolenyl-N¹,N⁴-di-(2-hydroxy-3-aminopropyl)-diaminobutane    (6);-   N¹,N²-dilinoleyl-N¹,N²-di-(2-hydroxy-3-aminopropyl)-diaminoethane    (7);-   N¹,N³-dilinoleyl-N¹,N³-di-(2-hydroxy-3-aminopropyl)-diaminopropane    (8);-   N¹,N⁵-dilinoleyl-N¹,N⁵-di-(2-hydroxy-3-aminopropyl)-diaminopentane    (9);-   N¹,N⁶-dilinoleyl-N¹,N⁶-di-(2-hydroxy-3-aminopropyl)-diaminohexane    (10);-   N¹,N⁸-dilinoleyl-N¹,N⁸-di-(2-hydroxy-3-aminopropyl)-diaminooctane    (11);-   N¹,N¹⁰-dilinoleyl-N¹,N¹⁰-di-(2-hydroxy-3-aminopropyl)-diaminodecane    (12);-   N¹,N¹²-dilinoleyl-N¹,N¹²-di-(2-hydroxy-3-aminopropyl)-diaminododecane    (13).

Example 7: Transfection with Inventive Lipids

Stock solutions of lipid in ethanol were prepared at concentrations ofbetween 5 mg/mL and 20 mg/mL and stored at −20° C. To performtransfections, nucleic acid was first diluted in DMEM (1 μg of nucleicacid in 50 μL of DMEM), then the desired amount of lipid stock solutionwas added. After adding the lipid, the solution was mixed thoroughly,and complexes were allowed to form for between about 5 minutes and about25 minutes before adding to cells. For the experiments depicted in FIG.1 through FIG. 6 , the lipid was used without the acetone purificationdescribed in Example 5.

FIG. 1 depicts transfection of primary human epidermal keratinocyteswith in vitro transcribed RNA encoding green fluorescent protein (GFP)complexed with the indicated lipids. As shown in the figure, cells weretransfected with high efficiency by DHDLinS.

FIG. 2 depicts a time course of the experiment of FIG. 1 , i.e.florescence measured at the indicated time points following transfectionusing DHDLinS. A fluorescent signal was detected one hour followingtransfection, and both the signal intensity and number of fluorescentcells increased for several hours following transfection.

FIG. 3 depicts transfection with various indicated amounts of RNA (innanograms) and lipid-to-RNA mass ratios (in micrograms of lipid permicrogram of RNA). As shown in the figure, all RNA amounts andlipid-to-RNA mass ratios tested yielded a fluorescent signal. Ingeneral, larger amounts of RNA yielded a stronger signal and/or largernumber of fluorescent cells, while minimal increase in fluorescencesignal was observed at lipid-to-RNA mass ratios greater than 5 μg/μg.

FIG. 4 depicts a transfection experiment with human peripheral bloodmononuclear cells (hPBMCs) instead of keratinocytes. As shown in thefigure, DHDLinS effectively transfected hPBMCs at both lipid-to-RNA massratios tested, while no transfection was observed with LIPOFECTAMINE3000, (cationic liposome formulation, “LF3000”) a commercialtransfection reagent.

FIG. 5A extends the transfection findings to a confluent layer ofprimary human epidermal keratinocytes instead of hPBMCs. As shown in thefigure, DHDLinS effectively transfected confluent primary humanepidermal keratinocytes at both lipid-to-RNA mass ratios tested, whilethe cells treated with LIPOFECTAMINE 3000 (cationic liposomeformulation) were not efficiently transfected. FIG. 5B depicts this athigher magnification. As shown in the figure, DHDLinS effectivelytransfected confluent primary human epidermal keratinocytes at bothlipid-to-RNA mass ratios tested, while the cells treated withLIPOFECTAMINE 3000 (cationic liposome formulation) were not efficientlytransfected.

FIG. 6A depicts the results of an experiment conducted as in FIG. 4 ,but with primary human adult dermal fibroblasts instead of hPBMCs. Asshown in the figure, DHDLinS effectively transfected primary human adultdermal fibroblasts at both lipid-to-RNA mass ratios tested, while thecells treated with LIPOFECTAMINE 3000 (cationic liposome formulation)were not efficiently transfected. FIG. 6B depicts this experiment athigher magnification. As shown in the figure, DHDLinS effectivelytransfected primary human adult dermal fibroblasts at both lipid-to-RNAmass ratios tested, while the cells treated with LIPOFECTAMINE 3000(cationic liposome formulation) were not efficiently transfected.

FIG. 7 depicts the results of an experiment conducted as in FIG. 3 , butwith DHDLinS purified by extraction with acetone as described in Example5. As shown in the figure, minimal increase in fluorescence signal wasobserved at lipid-to-RNA mass ratios greater than 2 μg/μg.

FIG. 8 depicts primary human epidermal keratinocytes cultured in a24-well plate, and transfected with 100 ng per well of in vitrotranscribed RNA encoding green fluorescent protein (GFP) complexed withthe compounds of Formula I, where n is as indicated or withLIPOFECTAMINE 2000 (cationic liposome formulation, “L2K”) orLIPOFECTAMINE 3000 (cationic liposome formulation, “L3K”). Images weretaken eight hours following transfection.

TABLE 1 Transfection with Inventive Lipids Transfection reagentFluorescence Intensity Formula I (n = 2) 6623 Formula I (n = 4), 8009a.k.a. “DHDLinS” Formula I (n = 5) 7554 Formula I (n = 6) 8596 Formula I(n = 8) 9170 Formula I (n = 10) 7842 Formula I (n = 12) 5631LIPOFECTAMINE 2000 3356 (cationic liposome formulation) LIPOFECTAMINE3000 3157 (cationic liposome formulation)

Table 1 depicts the results of an experiment in which 20,000 neonatalhuman epidermal keratinocytes (HEKn) per well of a 24-well plate weretransfected with 100 ng of in vitro transcribed RNA encoding greenfluorescent protein (GFP) complexed with the indicated lipids.Fluorescence was measured 24 hours after transfection. Numbers indicatemean fluorescence intensity per cell (a.u.).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

What is claimed is:
 1. A method for transfecting a cell with a nucleicacid, comprising contacting the cell with a nucleic acid and a compoundof the formula:


2. The method of claim 1, wherein the nucleic acid comprises DNA.
 3. Themethod of claim 1, wherein the nucleic acid comprises RNA.
 4. The methodof claim 1, wherein the nucleic acid is selected from an mRNA, an invitro transcribed mRNA, an siRNA, a miRNA, an lncRNA, an antisenseoligonucleotide, a ribozyme, a plasmid, a cosmid, a phage, a recombinantvirus, an episomal vector, an artificial chromosome, a yeast artificialchromosome, a mammalian artificial chromosome, an immune stimulatingnucleic acid, an antisense, an antagomir, an antimir, an in vitrotranscribed RNA, a microRNA mimic, a supermir, a U1 adaptor, an aptamer,and a synthetic RNA.
 5. The method of claim 1, wherein the nucleic acidis an in vitro transcribed mRNA.
 6. The method of claim 1, wherein thenucleic acid is an siRNA.
 7. The method of claim 1, wherein the nucleicacid is an antisense oligonucleotide.
 8. The method of claim 1, whereinthe nucleic acid is a plasmid.
 9. The method of claim 1, wherein thenucleic acid is an episomal vector.
 10. The method of claim 1, whereinthe nucleic acid is an immune stimulating nucleic acid.
 11. The methodof claim 5, wherein the in vitro transcribed mRNA comprises anon-canonical nucleotide.
 12. The method of claim 11, wherein thenon-canonical nucleotide is selected from 2-thiouridine, 5-azauridine,pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine,5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine,5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine,5-ethoxyuridine, 5-ethoxypseudouridine, 5-hydroxymethyluridine,5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine,5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine,5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine,5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine,4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine,4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine,4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine,4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine,4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine,pseudoisocytidine, N4-methylcytidine, N4-aminocytidine,N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine,5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine,5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine,5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine,5-methylpseudoisocytidine, 5-aminopseudoisocytidine,5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine,N4-methylpseudoisocytidine, 2-thio-5-azacytidine,2-thiopseudoisocytidine, 2-thio-N4-methylcytidine,2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine,2-thio-5-methylcytidine, 2-thio-5-aminocytidine,2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine,2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine,2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine,2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine,2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine,N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine,N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine,N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine,N4-methyl-5-aminopseudoisocytidine,N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine,N4-aminopseudoisocytidine, N4-amino-5-methylcytidine,N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine,N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine,N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine,N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine,N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine,N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine,N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine,N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine,N4-hydroxy-5-methylpseudoisocytidine,N4-hydroxy-5-aminopseudoisocytidine,N4-hydroxy-5-hydroxypseudoisocytidine,2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine,2-thio-N4-methyl-5-hydroxycytidine,2-thio-N4-methyl-5-methyl-5-azacytidine,2-thio-N4-methyl-5-amino-5-azacytidine,2-thio-N4-methyl-5-hydroxy-5-azacytidine,2-thio-N4-methyl-5-methylpseudoisocytidine,2-thio-N4-methyl-5-aminopseudoisocytidine,2-thio-N4-methyl-5-hydroxypseudoisocytidine,2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine,2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine,2-thio-N4-amino-5-hydroxycytidine,2-thio-N4-amino-5-methyl-5-azacytidine,2-thio-N4-amino-5-amino-5-azacytidine,2-thio-N4-amino-5-hydroxy-5-azacytidine,2-thio-N4-amino-5-methylpseudoisocytidine,2-thio-N4-amino-5-aminopseudoisocytidine,2-thio-N4-amino-5-hydroxypseudoisocytidine,2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine,2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine,2-thio-N4-hydroxy-5-hydroxycytidine,2-thio-N4-hydroxy-5-methyl-5-azacytidine,2-thio-N4-hydroxy-5-amino-5-azacytidine,2-thio-N4-hydroxy-5-hydroxy-5-azacytidine,2-thio-N4-hydroxy-5-methylpseudoisocytidine,2-thio-N4-hydroxy-5-aminopseudoisocytidine,2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine,N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine,8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine,7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine,N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine,N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine,N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine,N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine,8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine,7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.
 13. Themethod of claim 12, wherein the non-canonical nucleotide is5-methoxyuridine.
 14. The method of claim 1, wherein the nucleic acidand the compound are associated with a lipid aggregate.
 15. The methodof claim 14, wherein the lipid aggregate comprises a lipid nanoparticle.16. The method of claim 14, wherein the lipid aggregate comprises aliposome.
 17. The method of claim 14, wherein the lipid aggregatecomprises a lipid carrier.
 18. The method of claim 14, wherein thenucleic acid and the compound are associated with an additional lipid.19. The method of claim 18, wherein the additional lipid is selectedfrom dioleoylphosphatidylethanolamine (DOPE),1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol, and apolyethylene glycol (PEG)-modified lipid.